Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
FunctionMicrotubule plus-end tracking protein that promotes the stabilization of dynamic microtubules. Involved in the nucleation of noncentrosomal microtubules originating from the trans-Golgi network (TGN). Required for the polarization of the cytoplasmic microtubule arrays in migrating cells towards the leading edge of the cell. May act at the cell cortex to enhance the frequency of rescue of depolymerizing microtubules by attaching their plus-ends to cortical platforms composed of ERC1 and PHLDB2. This cortical microtubule stabilizing activity is regulated at least in part by phosphatidylinositol 3-kinase signaling. Also performs a similar stabilizing function at the kinetochore which is essential for the bipolar alignment of chromosomes on the mitotic spindle. Acts as a mediator of ERBB2-dependent stabilization of microtubules at the cell cortex.
Sequence similaritiesBelongs to the CLASP family. Contains 5 HEAT repeats.
DomainThe microtubule tip localization signal (MtLS) motif; mediates interaction with MAPRE1 and targeting to the growing microtubule plus ends.
Post-translational modificationsPhosphorylated by GSK3B. Phosphorylation by GSK3B may negatively regulate binding to microtubule lattices in lamella. Phosphorylated upon DNA damage, probably by ATM or ATR.
Cellular localizationCytoplasm > cytoskeleton. Cytoplasm > cytoskeleton > centrosome. Chromosome > centromere > kinetochore. Cytoplasm > cytoskeleton > spindle. Golgi apparatus. Golgi apparatus > trans-Golgi network. Cell membrane. Cell projection > ruffle membrane. Localizes to microtubule plus ends. Localizes to centrosomes, kinetochores and the mitotic spindle from prometaphase. Subsequently localizes to the spindle midzone from anaphase and to the midbody from telophase. In migrating cells localizes to the plus ends of microtubules within the cell body and to the entire microtubule lattice within the lamella. Localizes to the cell cortex and this requires ERC1 and PHLDB2. The MEMO1-RHOA-DIAPH1 signaling pathway controls localization of the phosophorylated form to the cell membrane.
Immunocytochemistry/ Immunofluorescence - Anti-CLASP2 antibody [KT68] (ab95373)Image is courtesy of an anonymous AbReview.
Immunocytochemical immunofluorescence analysis of formaldehyde-fixed SV-40 transformed bovine macrophages, labelling CLASP2 with ab95373 at a dilution of 1/100 incubated for 1 hour at 18°C in 10% FBS. Pereabilization was with 0.2% Triton X-100 for 10 minutes. Blocking used 10% serum incubated for 30 minutes at 18°C. Secondary was a Goat anti-rat polyclonal Alexa Fluor® 488 at 1/1000.
IHC image of CLASP2 staining in Mouse Brain formalin fixed paraffin embedded tissue section, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab95373, 1 µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
Western blot - Anti-CLASP2 antibody [KT68] (ab95373)Image is courtesy of an AbReview submitted by Kerry Woods.
All lanes : Anti-CLASP2 antibody [KT68] (ab95373) at 1/1000 dilution
Lane 1 : Human HEK 293 whole cell lysates Lane 2 : BoMAC SV-40 transformed bovine macrophage whole cell lysates
Lysates/proteins at 15 µg per lane.
Secondary Goat anti-rat polyclonal HRP conjugate at 1/2000 dilution Developed using the ECL technique
Performed under reducing conditions.
Predicted band size : 141 kDa Additional bands at : 65 kDa (possible non-specific binding).
Exposure time : 10 minutes
Image is courtesy of an AbReview submitted by Kerry Woods.
From the Left to right: lane 1, lane 2. Blocking was performed with 5% milk incubated for 30 minutes at 18°C.